The Xenopus protein kinase pEg2 associates with the centrosome in a cell cycle-dependent manner, binds to the spindle microtubules and is involved in bipolar mitotic spindle assembly.
By differential screening of a Xenopus laevis egg cDNA library, we have isolated a 2,111 bp cDNA which corresponds to a maternal mRNA specifically deadenylated after fertilisation. This cDNA, called Eg2, encodes a 407 amino acid protein kinase. The pEg2 sequence shows significant identity with members of a new protein kinase sub-family which includes Aurora from Drosophila and Ipl1 (increase in ploidy-1) from budding yeast, enzymes involved in centrosome migration and chromosome segregation, respectively. A single 46 kDa polypeptide, which corresponds to the deduced molecular mass of pEg2, is immunodetected in Xenopus oocyte and egg extracts, as well as in lysates of Xenopus XL2 cultured cells. In XL2 cells, pEg2 is immunodetected only in S, G2 and M phases of the cell cycle, where it always localises to the centrosomal region of the cell. In addition, pEg2 'invades' the microtubules at the poles of the mitotic spindle in metaphase and anaphase. Immunoelectron microscopy experiments show that pEg2 is located precisely around the pericentriolar material in prophase and on the spindle microtubules in anaphase. We also demonstrate that pEg2 binds directly to taxol stabilised microtubules in vitro. In addition, we show that the presence of microtubules during mitosis is not necessary for an association between pEg2 and the centrosome. Finally we show that a catalytically inactive pEg2 kinase stops the assembly of bipolar mitotic spindles in Xenopus egg extracts.
Roghi
C
C
Département de Biologie et Génétique du Développement, Université de Rennes I, Rennes, France.
Giet
R
R
Uzbekov
R
R
Morin
N
N
Chartrain
I
I
Le Guellec
R
R
Couturier
A
A
Dorée
M
M
Philippe
M
M
Prigent
C
C
D000595:Amino Acid Sequence; D000818:Animals; D064127:Aurora Kinases; D001483:Base Sequence; D002453:Cell Cycle / Q000502:physiology*; D018797:Cell Cycle Proteins; D002455:Cell Division / Q000502:physiology; D002460:Cell Line; D016764:Cell Polarity / Q000502:physiology*; D018385:Centrosome / Q000378:metabolism*; D047108:Embryonic Development; D005306:Fertilization / Q000502:physiology; D008870:Microtubules / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D011994:Recombinant Proteins / Q000378:metabolism; D008941:Spindle Apparatus / Q000648:ultrastructure*; D029867:Xenopus Proteins; D014982:Xenopus laevis
D018797:Cell Cycle Proteins; D011994:Recombinant Proteins; D029867:Xenopus Proteins; D011494:Protein Kinases; C112134:AURKA protein, Xenopus; D064127:Aurora Kinases; D017346:Protein Serine-Threonine Kinases